Ginkgo biloba is one of the oldest species in the world, and is considered a living fossil. Zhongliang, Chin. Pharm J. 1996, 31:(6) 326-331. It is indigenous to China, although now it has been introduced and cultured in most places in the world. The mature seeds are edible and are used as one of the traditional Chinese medicines. The chemical constituents of Ginkgo leaves are characterized by the presence of diterpene (sesquiterpene) lactones, and flavonoid glycosides as active principles, and ginkgolic acids as toxic substances in the Ginkgo biloba extract (GBE). Some of the above mentioned compounds are used as reference substances in qualitative and quantitative analysis of GBE. Ginkgolides A, B, C are diterpene lactones; bilobalide is a sesquiterpene lactone. The lactones are specific antagonists against platelet activating factors (PAF). Flavonoid glycosides are thought to have many useful biological activities including the activities of dilating coronary vessels, improving peripheral and brain blood circulation, and preventing intravascular thrombogenesis. For example, extracts from the leaves of the Ginkgo biloba tree (maidenhair tree) have been used for many years in the treatment of patients with conditions related to aging (DeFeudis, F. G., (1991) Ginkgo biloba extract. (EGb 761): Pharmacological activities and clinical applications. Editions Scientifiques Elsevier, Paris; Kleijnen, J., Knipschild, P. (1992) Lancet 340, 1136-1139). These conditions include cerebral insufficiency, which is defined by a group of symptoms exemplified by problems with short-term memory and concentration, lack of energy, tinnitis, headache, and depression (Kleijnen, J., Knipschild, P. (1992) Br. J. Clin. Pharmacol. 352-358). The most important active ingredients of the extracts are thought to be the flavonoids and the terpenoids, whereas ginkgolic acids are believed to cause contact dermatitis and other toxicities.
Ginkgo extracts are usually standardized in terms of their flavonoid glycoside and terpene lactone (ginkgolides and bilobalide) content (Sticher, O. (1993) Planta Med., 59, 2-11; Stinke, B., Muller, B., Wagner, H. (1993) Planta Med. 59, 155-160). Methods of preparing active extracts of Ginkgo biloba have been described in the art.
U.S. Pat. No. 5,637,302 is directed to a process where the lipophilic substances are removed from the extracts of Ginkgo biloba leaves using n-hexane, n-heptane or a solvent comprising a major proportion of toluene and a minor proportion of n-butanol. The resultant compositions (extracts) are disclosed as containing 22-26% flavone glycosides and 2.5-4.5 weight percent each of ginkgolides and bilobalide.
U.S. Pat. No. 5,512,286 is directed to extracts of Ginkgo biloba free of serum precipitating and/or hemagglutinating properties. The leaves are extracted using an aqueous acetone or alkanol (with 1-3 carbon atoms) or anhydrous methanol, precipitating out the lipophilic components, adding ammonium sulfate and extracting with methyl ethyl ketone, a multi-step extraction with water-immiscible butanol or pentanol (or, alternatively, with lead salts), alcohol extractions and column chromatography using a polyamide, or preferably a cross-linked polyvinyl pyrrolidone substitute.
Similarly, U.S. Pat. No. 5,399,348 describes extracts of Ginkgo biloba where the leaves are extracted using an aqueous acetone or alkanol (with 1-3 carbon atoms) or anhydrous methanol, precipitating out the lipophilic components, adding ammonium sulfate and extracting with methyl ethyl ketone, diluting in an aqueous alcohol solution which is treated with a lead salt or insoluble polyamide and extracting with an aliphatic or cycloaliphatic solvent.
EP-A 0 324 197 describes a method for the preparation of an extract from Ginkgo biloba leaves in which an aqueous solution of a lower alcohol or ketone, obtained after extraction of the leaves, is concentrated in the presence of kieselguhr. The resultant aqueous suspension is filtered through kieselguhr, the filtrate is extracted with butanone and the extract is freed from the solvent.
EP-A 330 567 relates to a method for the preparation of an extract from Ginkgo biloba leaves in which the crushed leaves are extracted with an aqueous ketone compound. This extract is concentrated until biflavones and hydrophobic compounds precipitate. After filtration the aqueous concentrate is rendered alkaline, whereby the proanthocyanidins precipitate. After separation of the precipitate and acidification of the filtrate, a liquid-liquid-extraction is carried out with a C.sub.4-6 -ketone compound in the presence of ammonium sulfate. The extract is obtained after stripping of the ketone compound.
The extracts from Ginkgo biloba leaves prepared by the methods in DE-B 17 67 098 and DE-B 21 17 429 are disclosed as being substantially free of alkylphenol compounds due to removal of the lipophilic components by a liquid-liquid extraction of the aqueous acetone extract with a substantially water-immiscible lipophilic solvent, e.g. with a chlorinated aliphatic lower hydrocarbon such as carbon tetrachloride. However, while the content of flavone glycosides is increased from 3 to 4% in the crude extract to approximately 24% in the final product, in this step, the therapeutically valuable ginkgolides and the bilobalide are considerably reduced so that their content in the final product in Example 1 of DE-B 21 17 429 is a maximum of 0.5% in the case of ginkgolides A, B, C, and J in total and approximately 0.3% in the case of bilobalide. Also, chlorinated aliphatic hydrocarbons are associated with certain toxicities and are generally undesirable.
U.S. Pat. No. 5,399,370 is directed to extracts of Ginkgo biloba having 40-60% flavone glycosides, 5.5-8.0% ginkgolides and 0.5-7.0% bilobalides. The method involves extraction with either aqueous alkanol or acetone or anhydrous methanol, precipitating out the lipophilic components, extracting with an ester of formic acid or acetic acid, and an additional extraction with a butanol or pentanol. Activated carbon may also be used to remove certain substances prior to the ethyl acetate or ester extraction.
The Ginkgo extract used most frequently at present for therapeutic purposes (tanakan.sup.R ; roekan.sup.R or tebonin.sup.R ; "EGb 761") contains, besides 24% flavone glycoside compounds, 6% terpene lactone compounds; see K. Drieu, La Presse Medicale Vol. 15 (1986), 1455-1457. These are the ginkgolides A, B, C and J as well as the bilobalide, which makes up approximately half of the 6%. The content of ginkgolide B found in currently available preparations ranges from about 0.88% to about 1.3%. The therapeutic daily dosage is 120 mg.
EP-A-86 315 describes a method of reducing the content of polymeric polyphenol compounds in the extracts by means of polyvinyl pyrrolidone in ethanolic-aqueous solution.
U.S. Pat. No. 4,981,688 discloses a method for extraction of Ginkgo biloba involving extraction from the leaves using an aqueous ketone solvent; concentration of the extraction liquors in order to precipitate the biflavonoids and the hydrophobic substances; alkalinization of the filtrate so as to precipitate the proanthocyanidins; acidification of the filtrate; liquid-liquid extraction of the filtrate with a C.sub.4 -C.sub.6 ketone in the presence of ammonium sulfate; and recovery of the extract by taking the ketone phase to dryness.
Flavones have also been extracted from Ginkgo biloba leaves with boiling water and an absorbent resin identified as being manufactured by Tianjing Gel Factory Model No. D1010. Xino et al., (1990) Chinese J. of Pharmaceuticals 21 (8): 340-341.
Other extracts of Ginkgo biloba and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,637,302; 5,700,468; 5,660,832; 5,158,770; 5,128,131; and 4,892,883.
Notwithstanding the numerous patents and publications concerning extractions of processes for Ginkgo biloba, there exists a need for processes which are free from toxic solvents, are relatively inexpensive and which further can be used to produce compositions with superior bioavailability.